Invasive aspergillosis (IA), caused by the fungus Aspergillus fumigatus, is associated with mortality rates of 40- 50%. In response to the lack of effective treatments, the Infectious Diseases Society of America highlighted A. fumigatus as one of only six pathogens for which it mandated that a substantive breakthrough is urgently needed. IA accounts for the largest financial burden of all invasive fungal infections, with an annual economic cost in the United States of over $1.1 billion. Driven by the growing immunosuppressed patient population, both the incidence and mortality due to A. fumigatus have risen three-fold in the last decade. While much is known regarding the cellular processes required for fungal pathogenesis, translating understanding into tangible clinical benefit has been difficult due to the fact that these fungal pathogens and their hosts have similar physiology. As a result, current antifungal agents have limited clinical efficacy, are poorly fungicidal in the host, are occasionally toxic, and are increasingly ineffective due to emerging resistance. Thus, innovative antifungal targeting agents and strategies are critically needed. It has been well established that molecules targeting fungal calcineurin (FC) have extremely potent antifungal activity against a broad range of fungi. Our collaborator at Duke University, Dr. William Steinbach, MD, has established that calcineurin is required for A. fumigatus hyphal growth and virulence. Moreover, calcineurin is required for fungal stress response and small molecule or genetic inhibition of calcineurin thwarts drug resistance. The challenge of exploiting FC as an antifungal agent is due to structural and sequence homology with human calcineurin (HC). Knowledge of the HC pathway and the immunosuppressive capacity of calcineurin inhibition has been one of the greatest contributions to our current solid organ and bone marrow transplantation abilities. However, inhibition of HC causes severe immunosuppression and toxicity. Recent chemical innovations have enabled Amplyx to rapidly create libraries of analogues of FK506 that were previously synthetically intractable to design and test specific FC inhibitors. Moreover, we have recently solved the X-ray structure of A. fumigatus calcineurin. allowing unprecedented structural insight to help guide our development efforts. Amplyx has established that inhibitors of FC are well tolerated in vivo at doses that provide a therapeutic benefit in an animal model of IA. To further develop this project we propose the following Aims: Aim 1. Design and synthesize a first generation library of 25 analogues of current hit compounds. Aim 2. Assess compounds for advancement employing in vitro studies. Aim 3. Characterize and select compounds for advancement employing in vivo studies.